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Abstract

Background—Structural damage during heart failure development leads to increased infiltration of leukocytes. Because purinergic signaling on immune cells may impact on the inflammatory response, we evaluated the role of ecto-5′-nucleotidase (CD73) on the development of heart failure after transverse aortic constriction (TAC) using global and T-cell–specific CD73−/− mice.

Methods and Results—Leukocytes infiltrating the failing heart were analyzed by a multistep enzymatic procedure over a period of 16 weeks using fluorescence-activated cell sorting. TAC significantly enhanced the infiltration of leukocytes, especially T cells. The fraction of CD73 expressing cells increased over time exclusively on cytotoxic T cells, T-helper cells, and regulatory T cells. Cardiac function significantly declined in T-cell–specific CD4-Cre+/−CD73flox/flox mice identical to that observed in global CD73 mutants and was associated with enhanced fibrosis (collagen, laminin, vimentin, periostin). Expression analysis by quantitative reverse transcription polymerase chain reaction of extracellular purine degrading enzymes and P1 and P2 receptors on T cells isolated from the injured heart revealed profound upregulation of the enzymatic machinery for hydrolysis of extracellular adenosine triphosphate and nicotinamide adenine dinucleotide, both pathways converging in the formation of AMP and adenosine via CD73. Among the P1 receptors, only the A2a receptor was significantly upregulated after TAC. T cells isolated from TAC-treated hearts show enhanced production of proinflammatory cytokines (interleukin-3, interleukin-6, interleukin-13, interleukin-17, macrophage inflammatory proteins-1α, and macrophage inflammatory proteins-1β) when CD73 was lacking.

Conclusions—Our data provide first evidence that CD73 on T cells plays an important anti-inflammatory role in TAC-induced heart failure, which is associated with antifibrotic activity and reduced production of proinflammatory cytokines most likely by activation of the adenosine A2a receptor.

Introduction

Experimental and clinical data suggest that heart failure is in part accompanied by inflammatory processes.1 Heart failure is one of the most prevalent and challenging chronic diseases, and pathological cardiac hypertrophy represents an important initial stage in the development of heart failure. Myocardial hypertrophy presents a gradual process involving cardiac dysfunction, upregulation of fetal gene expression patterns, reduced vascularization, changes in the composition of the extracellular matrix, and fibrosis.1,2

Elevated cytokine levels in circulating blood in patients experiencing heart failure suggest that heart failure is associated with local inflammatory processes.1 Proinflammatory cytokines are known to be involved in the development of pathological myocardial hypertrophy.1 Some studies revealed that patients with failing hearts are characterized by sustained activation of the innate immune system.3 Moreover, heart failure is accompanied by the expression of toll-like receptor 4, interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6.4 Although heart failure is generally known to be associated with chronic activation of the immune system, only little is known about infiltrating leukocytes and their function in promoting heart failure.

Extracellular generation of adenosine results from sequential dephosphorylation of extracellular adenosine triphosphate (ATP) to AMP by activity of CD39 (ectonucleoside triphosphate diphosphohydrolase), followed by ecto-5′-nucleotidase CD73. There is increasing evidence that adenosine formed by CD73 plays a key role in the regulation of inflammatory processes by modulation of endothelial adhesion, transmigration, and activation of T cells.5 Whereas adenosine triphosphate primarily acts as a proinflammatory signal for P2 receptors, its metabolite, adenosine, leads to both pro- and anti-inflammatory effects by activation of P1 receptors depending on the receptor subtype.6 The purine nucleoside adenosine is involved in a variety of physiological and pathophysiological processes and has been reported to display tissue protective properties in ischemia and inflammation.7

The aim of this study was the analysis of functional and immune cell-specific parameters related to the expression of CD39, CD73 and pannexin 1 (Panx1), adenosine receptors A1, A2a, A2b, and A3, and additional members of the purinergic signaling cascade on infiltrating leukocytes in failing hearts after transverse aortic constriction. We used a global and T-cell–specific CD73−/− mutant to delineate the cellular production and function of adenosine in the pathogenesis of heart failure.

Methods

Mice

Animal experiments were performed in accordance with the national guidelines on animal care and were approved by the Landesamt für Natur, Umwelt, und Verbraucherschutz (LANUV, Nordrhein-Westfalen, Germany). We used different mouse strains: CD73−/− mutant mice (CD73−/− or CD73 knockout [KO]), T-cell–specific CD73−/− mutant mice (CD4-Cre+/− CD73flox/flox or CD4 CD73 KO), and the corresponding control animals CD4-Cre−/−CD73flox/flox and c57Bl/6 (wild type [WT]). All animals were female mice and ranged from 20 to 25 g in body weight and aged between 8 and 12 weeks. All animals used in this study were bred and kept at the central animal research faculty of the Heinrich-Heine-University, Düsseldorf, Germany. They were fed with a standard chow diet and received tap water ad libitum. The mouse strains were generated in our laboratory as previously described in detail by Koszalka et al8 and are based on c57Bl/6 background. Animals homozygous for floxed CD73 and hemizygous for Cre-recombinase were used as T-cell–specific CD73−/− mice. Genotypic characterization of mice was performed using routine polymerase chain reaction (PCR) and the following primers: CD73-A GTAGTGCTCGGAGCACGTACA-3′; CD73-B 5′-CAGTGTGAGGCACAGAGCATCTCA-3′; Cre-A 5′-CGATGCAACGAGTGATGAGG-3′; Cre-B 5′-GCATTGCTGTCACTTGGTCGT-3′.

Surgical Procedures

Twelve-week-old adult mice were anesthetized with a mixture of 1.5 to 2.0 vol% isoflurane and buprenorphine (0.05–0.1 mg/kg, subcutaneous injection). After endotracheal intubation and mechanical ventilation at a rate of 150 to 180 strokes/min and a tidal volume ranging from 220 to 280µL, animals were placed in a supine position on a warming pad. Under an upright dissecting microscope (Leica MS05), the aortic arch was exposed and constricted by tying a 7-0 silk ligature around a wire with a diameter of 0.36 mm. The wire was removed, resulting in a reproducible transverse aortic constriction (TAC) of ≈80%.

Echocardiography

Functional variables were assessed by echocardiography using a high-frequency, high-resolution digital imaging platform for small animals with an 18 to 38 MHz transducer MS400 (Vevo2100; Visual Sonics, Canada). The standardized examination was performed in parasternal long-axis view, short-axis view, 4-chamber view, and aortic arch view. Functional variables were calculated from B- and M-Mode in parasternal long-axis view, short-axis view, and modified Simpson rule.

Tissue Digestion, Cell Isolation, and Flow Cytometry

Techniques used for digestion of the heart, isolation of immune cells, and flow cytometry have been performed as previously described.9 A brief description is given in the Data Supplement and Table I in the Data Supplement.

Detailed information of RNA isolation, reverse transcription, and preamplification are given in the Data Supplement. Using TaqMan Gene Expression Master Mix and TaqMan Gene Expression Assay on the corresponding TaqMan Array Micro Fluidic Cards (Applied Biosystems), the expression of the following target genes of purinergic signaling pathways was determined by quantitative reverse transcription PCR (see Table II in the Data Supplement): genes of the adenosine receptors (A1, A2a, A2b, A3), ADP and ATP receptors (P2X1, 4, 5, 7, and P2Y1, 2, 4, 6), ectoenzymes (CD73, CD39, CD38, ENPP1, ENPP3), transporters and channels for nucleosides and nucleotides (Panx1, connexin 37 [Cx37], connexin 43 [Cx43], Cnt2, Ent1, Ent2), and 3 endogenous expression controls (β-actin, 18S, and Tbp). All kits were used according to the manufacturer’s protocols. Each sample produced 3 technical replicates, resulting in a mean per sample. Each microfluidic card was measured by a ViiA 7 Real-time PCR system (Life Technologies). The analysis was performed with ExpressionSuite Software v1.0.2 (Life Technologies). To compare gene expression between different samples, ΔCt method was used; 18S was used as endogenous control. To quantify the relative expression of the target gene in comparison with the endogenous control, relative expression is depicted as 2ΔCt-value. Moreover, we added experiments to evaluate the differentiation and activation of T cells. For this purpose, we measured the expression of t-bet (marker of Th1-cell differentiation), GATA3 (marker of Th2-cell differentiation), Rorγt (marker of Th17-cell differentiation), FoxP3 (marker of differentiation of regulatory T cells), CD69 (activation marker of T cells), and CD44 (marker of memory T cells) in T cells after 4 weeks.

Histology and Immunohistology

For histological analysis, hearts were rapidly excised, freed from blood by retrograde perfusion with modified Krebs–Henseleit buffer according to Langendorff for a few seconds, embedded in embedding medium, and frozen in prechilled liquid methylbutane at −40°C. Samples were stored at −80°C or at −20°C before cutting. The hearts were cut in 8 μm slices from apical to basal in 4 (basal heart) or 5 (4 weeks after TAC) spots 1 mm apart from each other, 6 slices each spot. Because T-cell infiltration, upregulation of CD73, heart weight, and hemodynamic variables reached almost constant steady-state values, we have chosen 4 weeks as time point of the chronic phase to measure fibrosis. Staining was performed with picrosirius red according to manufacturer’s protocols. The extent of fibrosis per heart was determined in 6 high-power fields at ×20 magnification in each spot using ImageJ. The image acquisition was operated by an Olympus MX 61 microscope.

The extent of apoptosis was assessed in the abovementioned spots by CardioTACS TdT In Situ Apoptosis Detection Kits (Trevigen Inc, Helgerman Ct, Gaithersburg) according to manufacturer’s instructions. Apoptotic cells were measured in 6 high-power fields at ×20 magnification in each spot and depicted as mean percentage of apoptotic cells of total cell count in all spots per heart. For immunohistology, we used antibodies against α-actinin (anti-mouse) and cardiac troponin T (anti-mouse) to stain cardiomyocytes, against the extracellular matrix proteins laminin (anti-rabbit) and vimentin (anti-rabbit), against periostin (anti-rabbit) as a marker of activated fibroblasts and the injury-induced extracellular molecule Tenascin C (anti-mouse) in 6-µm-thin midventricular slides. Quantification was performed in 4 regions per sample at ×10 magnification.

Statistics

Statistical analysis was performed using Microsoft Excel and SPSS (IBM). All data are illustrated as mean±SD or SEM as indicated. To determine significant differences between groups, ANOVA and post hoc analysis, including Bonferroni, Tukey-Kramer, and Dunnett or Dunnett-C test, have been applied. A linear mixed-effects model with unstructured covariance matrix was applied to echocardiographic data. P≤0.05 has been considered being statistically significant.

Serial assessment of functional variables by echocardiography for 8 weeks after induction of TAC revealed a significant impairment of ejection fraction together with an increase in end-diastolic and end-systolic volume in global CD73−/− mice compared with WT and CD4-Cre−/−CD73flox/flox controls (Table III in the Data Supplement). WT and CD4-Cre−/−CD73flox/flox showed similar functional variables at all time points. Surprisingly, functional deterioration was similar in time course and extent in T-cell–specific CD73−/− mutants. As shown in Figure 3, global CD73−/− and T-cell–specific CD73−/− mice showed a similar reduction in ejection fraction. A complete overview of echocardiographic variables is given in Table III in the Data Supplement. As shown in Figure 3D, heart weight increased after TAC as expected, reaching stable values after 4 weeks. We found that global and T-cell–specific deletion of CD73 did not influence the TAC-induced increase in heart weight (Figure 3E), suggesting that lack of CD73 did not alter hypertrophy induction. Similarly, analysis of heart weight 8 weeks after TAC revealed no significant differences between WT and CD4 CD73 KO (data not shown).

Functional variables assessed by echocardiography in control animals (c57Bl/6), CD73 knockout (ko) mice (CD73−/−), and T-cell–specific CD73 ko mice (CD4-Cre+/− CD73flox/flox). A, Ejection fraction in [% ± SD] from baseline to 8 weeks after pressure overload. Although sham animals showed a constant ejection fraction over time (not shown) comparable with baseline conditions in control mice, ejection fraction decreased consistently in all mice strains after transverse aortic constriction, showing significant differences in ko mice compared with control mice (n=8–12; *P<0.05; **P<0.01; ***P<0.001). Comparable functional impairment of ejection fraction was increased in both knockout strains, associated by increasing end-systolic (B) and end-diastolic (C) volume (both in µL±SD). D, Increase in heart weight over time in wild type (WT; n=4–9). At all time points, heart weight is significantly different from basal conditions, reaching maximal values after 4 weeks. E, Increase in heart weight is similar between the mice strains after 7 days after transverse aortic constriction compared with basal conditions.

Functional deterioration of the heart was paralleled by significantly increased interstitial fibrosis when CD73 was lacking on T cells (Figure 4A). Measurements were performed 4 weeks after TAC because leukocyte infiltration (Figure 1) and CD73 expression on T cells (Figure 2) reached stable values, indicating a chronic phase. We found the extracellular matrix proteins laminin (Figure 4B) and vimentin (Figure 4C) to be significantly elevated, as well as periostin (Figure 4D), a marker for activated fibroblasts. Periostin-positive cells were regularly associated in clusters with Tenascin C, another extracellular matrix protein, but did not fully colocalize (Figure 4D). We also found the extent of apoptosis to be significantly increased in hearts of T-cell–specific CD73−/− mice (Figure 4E).

To further study the underlying mechanism of the impaired ejection fraction when CD73 was lacking, CD45+CD3+ T cells were isolated from the injured heart 4 weeks after TAC, then fluorescence-activated cell sorting (FACS) sorted and the expression of various ectoenzymes, adenosine and ATP receptors, and nucleotide/nucleoside transporters were measured by reverse transcription PCR. Values obtained were compared with corresponding measurements from circulating T cells. From the data summarized in Figure 5, it can be seen that in WT mice CD73, CD39, ENPP1/ENPP3 (pyrophosphatases), as well as CD38, were significantly increased. Similar changes were observed on T cells from T-cell-CD73−/− mice.

Among the 4 adenosine receptors, the A2a adenosine receptor (A2AR) was strongly upregulated on T cells when migrating into the injured tissue of WT mice (Figure 5B); in mice lacking CD73 on T cells, this increase was significantly higher. The expression of the A1R, A2bR, and A3R was generally low, and there were no major TAC-induced changes. Among the ATP receptors, the P2X7 and P2Y3 were upregulated on migration into the TAC-injured heart; however, changes were similar in WT- and T-cell-CD73−/− mice.

Analysis of channels for ATP and ADP, such as Panx1, Cx37, and Cx43, as well as the equilibrative nucleoside transporter 1 (ENT1), were found to be significantly increased on infiltrating T cells (Figure 5C). Although there were no major differences between WT and T-cell-CD73−/− mice, only Panx1 reached the level of significance in mice lacking CD73 (Figure 5C).

A, Cytokine secretion profile in pg/mL±SD depicted per 10 000 CD45+ CD3+ stimulated T cells (n=3 in wild type [WT], n=4 in CD4-Cre+/− CD73flox/flox). The ordinate on the right side of the graph is indicating the scale for measured concentrations of interleukin (IL)-3 and IL-6 (pg/mL). B–G, Expression of differentiation and activation markers of circulation T cells (blood) and infiltrating T cells (heart) 4 weeks after TAC. *P<0.05; **P<0.01.

In view of the observed CD73-dependent cytokine pattern, we investigated whether a specific T-cell population is predominantly influenced by CD73. As summarized in Figure 6B through 6E, we found that the upregulation of the T-cell lineage-specific transcription factors t-bet (Th1 cells), GATA3 (Th2 cells), Rorγt (Th17 cells), and FoxP3 (T reg cells) within infiltrating T cells was similar in WT and KO mice when compared with circulating T cells. Interestingly, the expression of CD44, a marker for memory T cells, was significantly enhanced both in circulating and in infiltrating T cells only when CD73 was lacking (Figure 6F), whereas the early activation marker CD69 showed a similar tendency without reaching the level of significance (Figure 6G).

Discussion

The ectoenzyme CD73, expressed on many cell types, such as immune cells, endothelial cells, and mesenchymal cells, is generally assumed to produce anti-inflammatory adenosine acting on A2a receptors.6 The present study demonstrates that mice lacking CD73 solely on T cells respond to TAC-induced heart failure with further deterioration of contractile function and increased cardiac fibrosis accompanied by more activated fibroblasts in the myocardium. This phenotype fully parallels the functional impairment observed in the global CD73 mutant (Figure 3).10 As to the underlying mechanism, we provide evidence that the adenosine production on T cells migrating into the injured heart is enhanced by activation of extracellular nucleotide release or degradation and that adenosine formed by T cells critically controls proinflammatory cytokine production. This places CD73-derived adenosine on T cells into a crucial position in the healing process of TAC-induced cardiac injury and may prevent maladaptive tissue remodeling.

Inflammation in the myocardium of failing hearts to date was mainly characterized by immunohistochemistry including data of human endomyocardial biopsies.11 Collectively, these semiquantitative findings show chronic low-grade inflammation and more abundant presence of CD3-positive lymphocytes, CD68-positive macrophages, and CD11a/CD18-positive cells and macrophages expressing TNF-α.11 To our knowledge, the present study is the first to document in an experimental model of heart failure the kinetics of immune cell infiltration over a period of 16 weeks using fluorescence-activated cell sorting. Apparently, 2 phases can be differentiated in the TAC model: an initial phase with transient increase of granulocytes, monocytes, and B cells, followed by a second chronic phase associated with increased cytotoxic T cells, T-helper cells, and T reg cells. The initial inflammatory boost within the first week after TAC is most likely because of tissue injury in response to the initiation of TAC. However, there is also chronic cardiomyocytes death as judged by the increased rate of apoptosis (Figure 4E), which is likely to maintain the T-cell–associated inflammation.

Several lines of experimental evidence suggest that CD73 on T cells plays a crucial role in the chronic phase of ventricular remodeling after TAC-induced cardiac injury. Not only were T cells the only immune cell subfraction, which remained elevated over time, but also the expression of CD73 significantly increased only on measured T-cell subtypes within the first 4 weeks after TAC and thereafter remained elevated. Finally, the observed deterioration of contractile parameters in T-cell CD73−/− mutants was identical to global CD73−/− mice, suggesting that the adenosine production on T cells by CD73 was responsible for the observed phenotype. Interestingly, the expression of CD73 on B cells, natural killer cells, granulocytes, and monocytes remained unchanged under these conditions and was generally lower than that of T cells. Because global and T-cell–specific CD73−/− mice were functionally identical after TAC, this strongly suggests that the adenosine production by these latter immune cells has a major impact in the remodeling phase. Still unresolved is the question which T-cell subtype conferred the cardioprotective action and in particular what role T reg cells, known to promote tissue repair,12 play in this process.

Naive lymphocytes circulating in the body are in a resting state but become activated on recognition of damage antigens formed by the injured heart, resulting in sterile inflammation.13 Recent evidence suggests that T cells in this process of activation undergo important metabolic reprogramming involving signal transduction and epigenetic programming.14 In the present study, we found that T cells migrating into the injured heart strongly upregulate the expression of channels responsible for the release of nucleotides (Panx1, Cx37, Cx47), as well as ectoenzymes responsible for the hydrolysis of ATP to adenosine (CD39, CD73, ENPP 1+3). Controlled release of ATP through Panx115 and connexin hemichannels16 from activated cells has been reported. Rapid activation of mitochondrial ATP production fuels ATP release from activated T cells.17

The binding of ATP, considered to act as a danger signal, to P2X7 receptors enhances inflammation and tissue injury via Nlrp3 inflammasome activation.18 In our study, we found T-cell P2X7 within the heart to be upregulated compared with circulating T cells, supporting the hypothesis that T-cell–derived ATP may participate in triggering inflammation in heart failure. It should be kept in mind that the extracellular concentration of ATP is controlled not only by its rate of production but also by the activity of enzymes degrading ATP. We found that CD39 on activated T cells was massively upregulated, and this was associated with significant upregulation of the pyrophosphatases ENPP1 and ENPP3 breaking down ATP to AMP and pyrophosphate. In functional terms, this suggests that the biological half-life of proinflammatory ATP is reduced, which, together with the observed upregulation of CD73, results in the enhanced formation of anti-inflammatory adenosine. It should be noted that we also observed upregulation of CD38, a nicotinamide adenine dinucleotide+ nucleosidase, which degrades extracellular nicotinamide adenine dinucleotide, reported to be released from T cells19 via ENPP1 to AMP and adenosine.20

The hypothesis of a decreased anti-inflammatory feedback in mice lacking CD73 on T cells is supported by our finding that T cells devoid of CD73 show enhanced secretion of cytokines of the Th17 panel (IL-6, IL-10, IL-17) but also secrete significantly more IL-3, IL-13, MIP-1α, and MIP-1β. Recent studies suggest that the failing human heart is a source of proinflammatory cytokines.21 Inflammatory biomarkers, such as C-reactive protein and inflammatory mediators (TNF-α and IL-6, eg), increase systemically in heart failure associated with disease progression.20 IL-6 can upregulate antiapoptotic Bcl-2 and downregulate proapoptotic Bax in cardiomyocytes and is known to play an important role in pressure overload and congestive heart failure.22 The observed enhanced rate of apoptosis may be a consequence of elevated IL-6 formation. However, IL-10, a potent anti-inflammatory cytokine, is known to inhibit infiltration of monocytes or macrophages into the injured tissue and was described to attenuate pressure overload–induced hypertrophic remodeling via STAT3-dependent inhibition of nuclear factor-κB.23,24 Similarly, IL-13, aside stimulating B-cell production and differentiation, inhibits the activation of macrophages.23 IL-17 is well known to play a key role in inflammatory processes and is likely to be involved in the enhanced cardiac fibrosis when adenosine is lacking. MIP-1β was reported to be secreted by cytotoxic T cells25; when acting locally, it can bind to vascular cell adhesion molecule-1 on endothelial cells in the initial step of leukocyte recruitment to the inflammatory site. Although both MIP-1α and MIP-1β are potent chemoattractants for monocytes, macrophages, and dendritic cells, they differ in their preferred target T cells. Although MIP-1α preferentially attracts CD8+ T cells, CD4+ T cells are more responsive to MIP-1β. In summary, our data support the hypothesis that adenosine generated by CD73 on T cells acts through cytokines to limit local inflammation in an autocrine and paracrine fashion.

Analysis of transcription factors typical for differentiation into T-cell subpopulations revealed a similar upregulation after infiltration, whereas the expression of CD44 was significantly enhanced both in circulating and in infiltrating T cells under these conditions (Figure 6F). This suggests that lack of the anti-inflammatory loop via CD73 does not influence the polarization of T cells infiltrating the injured myocardium, but result in a global increase in memory T cells (CD44) maintaining proinflammatory conditions. This process might be involved in the adverse remodeling process characterized by activated fibroblasts and increased fibrosis.

The anti-inflammatory effects of adenosine are generally mediated by the A2AR, which plays a critical role in the pathophysiological modulation of inflammatory responses in many experimental models.26 A2AR deficiency in T regs was reported to reduce their immunosuppressive efficacy in vivo.27 Here, we found that among the 4 adenosine receptors, only the A2AR was strongly upregulated on T cells when activated within the injured heart. Upregulation of the A2AR on immune cells within inflamed tissue has been observed in an arthritis model28 and is generally interpreted as the body’s own response to limit inflammation. If CD73 is lacking on immune cells, less adenosine is formed by the heart,29 and this is likely to cause an enhanced inflammatory response, despite compensatory upregulation of the high expression of the A2AR on T cells observed in the present study. This latter observation opens the interesting possibility that stimulation of the failing heart with selective A2AR agonists may be therapeutically beneficial to limit the inflammatory response and to reverse adverse cardiac remodeling. Potent and selective A2AR agonists have been synthesized.6 However, A2AR agonists are also potent vasodilators,30 which may limit their clinical utility. To differentiate between the vasodilatory and the immunosuppressive action of A2AR activation, phosphorylated A2AR agonists (prodrug) have been synthesized, which act through CD73-mediated local degradation to the active agonist.28 Also note that the A2AR on fibroblasts is a fine-tune regulator of the collagen balance (collagen1/collagen3) in scar tissue.31

In summary, our data show that CD73 on T cells plays a critical role in TAC-induced heart failure, in that T-cell–derived adenosine reduces the production of proinflammatory cytokines, thereby limiting the inflammatory response by paracrine and autocrine feedback mechanisms. Metabolic remodeling accelerates the release of ATP from activated T cells, which is associated with enhanced degradation to adenosine. Activated T cells also significantly upregulate the expression of the A2AR, which seems to be a promising target to attenuate adverse cardiac remodeling.

Acknowledgments

We thank Katharina Raba from the Core Flow Cytometry Facility at the Institute for Transplantation Diagnostics and Cell Therapeutics at the University Hospital Düsseldorf for technical assistance.

Sources of Funding

This work was supported by the German Heart Foundation/German Heart Research Foundation (DSHF) (F/43/12 to C.Q.).

CLINICAL PERSPECTIVE

Structural damage of the heart during the development of heart failure leads to increased infiltration of leukocytes, which is likely to impact the severity of the disease. Here, we identified upregulation of enzymes involved in the extracellular hydrolysis of adenosine triphosphate/nicotinamide adenine dinucleotide and the enhanced expression of the A2a receptor on T cells as the body’s defense system fighting inflammation of the failing heart. Thus, targeting purinergic signaling on T cells may constitute a promising approach to reduce injury-induced inflammation. This can be accomplished by further activating CD73 pharmacologically or using specific A2a receptor agonists. Both interventions are likely to reduce cardiac inflammation and improve cardiac function of the failing heart.